DNA sequencing

ABSTRACT

To sequence DNA, DNA samples marked with fluorescent infrared dye are applied at a plurality of locations for electrophoresing in a plurality of channels through a gel electrophoresis slab. The channels are scanned with a laser and a sensor, that include a microscope focused on the gel slab. The focal point and slab are adjusted with respect to each other so that the focal point of the microscope remains on the gel slab during a scan. The data from the scan is directly used to amplitude modulate density readings on a display, and the scan is displayed in a horizontal sweep of a cathode ray tube, whereby said cathode ray tube provides intensity displays of bands representing DNA. Different sizes of glass gel sandwiches may be mounted to the same console for different sequencing tasks.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of applicationNo. 07/763,230 filed Sep. 20, 1991, now U.S. Pat. No. 5,230,781 and ofapplication No. 07/570,503 filed Aug. 21, 1990, now U.S. Pat. No.5,207,880 which are continuations in part of U.S. Pat. No. 07/078,279filed Jul. 27, 1987 now abandoned, which is a division of U.S.application No. 594,676 for DNA SEQUENCING filed by Middendorf et al. onMar. 29, 1984, and assigned to the same assignee as this application,now U.S. Pat. No. 4,729,947.

BACKGROUND OF THE INVENTION

This invention relates to the sequencing of DNA strands.

In one class of techniques for sequencing DNA, identical strands of DNAare marked. The strands are separated into four aliquots. The strands ina given aliquot are either individually cleaved at or synthesized to anybase belonging to only one of the four base types, which are adenine,guanine, cytosine and thymine (hereinafter A, G, C and T). The adenine-,guanine-, cytosine- and thymine-terminated strands are thenelectrophoresed for separation. The rate of electrophoresis indicatesthe DNA sequence.

In a prior art sequencing technique of this class, the DNA strands aremarked with a radioactive marker, and after being separated byelectrophoresis, film is exposed to the gel and developed to indicatethe sequence of the bands. The range of lengths and resolution of thistype of static detection is limited by the size of the apparatus.

It is also known in the prior art to use fluorescent markers for markingproteins and to pulse the fluorescent markers with light to receive anindication of the presence of a particular protein from thefluorescence.

The prior art techniques for DNA sequencing have several disadvantagessuch as: (1) they are relatively slow; (2) they are at least partlymanual; and (3) they are limited to relatively short strands of DNA.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a noveltechnique for DNA sequencing.

It is a still further object of the invention to provide novelapparatuses and methods for sequencing relatively large fragments ofDNA.

It is a still further object of the invention to provide novelapparatuses and methods for sequencing DNA fragments of 100 bases ormore.

It is a still further object of the invention to provide a technique forcontinuous sequencing of DNA.

It is a still further object of the invention to continuously sequenceDNA without the spatial limitations of range of lengths and resolution.

It is a still further object of the invention to provide a noveltechnique for continuously sequencing DNA using fluorescent detection.

It is a still further object of the invention to provide a noveltechnique for DNA sequencing using a fluorescent marker fastened to theDNA, or the inherent fluorescence of the DNA itself.

It is a still further object of the invention to provide a noveltechnique for continuously sequencing DNA marked with fluorescence whichmore clearly distinguishes marked DNA fragments from backgroundfluorescence.

It is a still further object of the invention to provide a noveltechnique for scanning fluorescent material.

It is a still further object of the invention to provide a noveltechnique for displaying fluorescent material.

In accordance with the above and further objects of the invention,strands of DNA are continuously electrophoresed and identified. For thispurpose, the strands are fluorescently marked by direct labelling offluorescent markers to the strands or by fluorescently labelled probeshybridized to the separated strands. The light emitted while irradiatingthe strands near the terminal end of the electrophoresis channel isdetected and correlated. The electrophoresis conditions are selected sothat strands being electrophoresed near the terminal end of theelectrophoresis channel are fully resolved prior to the resolution oflonger strands which are at the entrance end of the electrophoresischannel, and so on, in a continuous process over a period of time.

The apparatus for such continuously sequencing of DNA includes one ormore electrophoresis channels, each adapted to receive fluorescentlylabeled DNA strands, having at one end a base of a given type. Each ofthe channels has a path and electrical field across it identical in itscharacteristics to the path of the other channels and electrical fieldsacross the other channels.

To provide marking, either a fluorescent marker is attached to the DNAfragments prior to their being electrophoresed, or probes are used tocombine or hybridize with the DNA strands. In the latter case, thedetection is accomplished by detecting a fluorescent marker that ischemically attached to the probe. In the preferred embodiment, themarker is a dye that fluoresces in the infrared or near infrared region.

The electrophoresis may be provided in conventional gel slabs or in tubegels such as gel filled capillary tubes or buffer filled capillarytubes. For the configuration using conventional gel slabs, oneembodiment provides for a different input section for each of fourchannels that are for a corresponding one of the A, G, T and C strands.Other embodiments allow for less than four gel channels by judiciouslycombining one or more base types A, G, T, or C in a channel. The strandsare detected during electrophoresis either in the gel by scanning backand forth across the gel at a fixed distance from the entrance end ofthe gel or by one or more fixed detectors located at a fixed distancefrom the entrance end of the gel or after leaving the gel. The strandsare detected in a manner that indicates their mobility in the gel toindicate the sequence of the A, G, C and T strands of different lengths.

The detection apparatus includes a light source, such as a laser or arclamp or other suitable source that emits light in the optimum absorptionspectrum of the marker. The light may be split by the use of fiber. Inthe preferred embodiment, the light source is a diode laser thatirradiates the channels with near infrared or infrared light having awavelength that matches the absorbance region of the marker. Thedetector includes a light sensor which is preferably an avalanchephotodiode sensitive to the near infrared or infrared light emission ofthe marker. It may include a filtering system having a pass bandsuitable for passing selectively the optimum emission of the fluorescentmarker to the light sensor. Correlation of the channel in which thefluorescent light is detected and the time of detection of thefluorescent light indicates: (1) if the type of base termination ornucleotide cleavage is A, G, C or T or a combination thereof for thoseembodiments which have more than one base type in a channel; and (2) thetime sequence of separation of each strand in each channel of theelectrophoresis gel. This information, in turn, indicates the overallsequence of strands.

To use the apparatus to sequence DNA strands, identical DNA strands arenormally formed of a length greater than 100 bases. In one embodiment,the strands are marked by a suitable marker at one end. The strands aredivided into four aliquots and the strands within each aliquot arecleaved at any base belonging to one or more specific base types. Inanother embodiment, strands are synthesized to any base belonging to aspecific base type. These four aliquots are then electrophoresed throughone or more identical channels to separate strands so that the shorterstrands are resolved towards the end of the gel prior to resolution ofthe longer strands, which may be near the entrance end of the gel at thetime the shorter strands are being resolved. This occurs in a continuousprocess so a substantial number of different length strands may beresolved in a relatively short gel. This methodology takes advantage oftime-resolved bands, as opposed to the limitations of spatial-resolvedbands.

The gel size, electric field and DNA mobilities are such that the moremobile bands are fully resolved while the less mobile bands are yetunresolved in a continuous process such that at least ten percent of thebands have been resolved by electrophoresis in the gel while the lessmobile bands which are near the entrance end of the gel are not fullyresolved. These less mobile bands become resolved little by little overtime in a continuous fashion without interruption of the movement ofthese bands through the gel. The markers are detected by transmittinginfrared light to fluorescently marked DNA strands.

To obtain maximum information in those embodiments in which a gel slabis scanned, a microscope and laser are moved together on a platform withrespect to the gel. To ensure parallelism between the microscope/laserassembly and the slab gel as well as optimal focusing of the assemblyonto the slab gel, in one embodiment, the sensor determines the point inthe glass-gel-glass sandwich having minimal fluorescence and focuses onit. This minimal flourescence is due to the reduced flourescence of thegel as compared to the glass. The microscope is continually moved andrefocused as scanning takes place to maintain the optimum focus.

In another scanning embodiment, the microscope determines the optimalfocus position at one location on the glass-gel-glass sandwich and thenis moved to another location on the glass-gel-glass sandwich where anoptimal focus position is determined for that location. The scanningmechanism is then pivoted so that the scanning mechanism is parallel toa line connecting the two optimal focus positions previously determined.This insures that the gel slab is in the focal plane of themicroscope/laser assembly. The intensity signal received from thescanning microscope/laser assembly which indicates the presence orabsence of DNA strands is directly transmitted to the intensity input ofthe computer monitor so that the display varies in brightness ratherthan providing an amplitude trace.

For purposes of focusing, either by pivoting the scanning mechanism orby adjusting the microscope at different points during a scan to followan established line, the microscope/laser assembly moves orthogonal tothe plane of the glass-gel-glass sandwich at one end of the scan inorder to locate the position of lowest flourescence, which is theposition of the gel slab between the two glass plates. Then it moves tothe other end of its scan and performs the same function. These twofocusing movements are utilized to move the scan mechanism in oneembodiment and to program the movement of the microscope focus inanother embodiment so that the microscope in the one embodiment movescontinuously along a single line and the scan mechanism has beenprepositioned such that that line is parallel to the gel slab, and inthe other embodiment, the focus is changed at six points to accommodatea non-parallel alignment between the scan mechanism and the gel.

From the above summary, it can be understood that the sequencingtechniques of this invention have several advantages, such as: (1) theytake advantage of resolution over time, as opposed to space; (2) theyare continuous; (3) they are automatic; (4) they are capable ofsequencing or identifying markers in relatively long strands includingstrands of more than 100 bases; (5) they are relatively economical andeasy to use; (6) they permit efficient focusing of a light sensor ontothe DNA bands; and (7) they provide an easy to observe display.

SUMMARY OF THE DRAWINGS

The above noted and other features of the invention will be betterunderstood from the following detailed description when considered withreference to the accompanying drawings in which:

FIG. 1 is a block diagram of an embodiment of the invention;

FIG. 2 is a perspective view of a portion of the embodiment of FIG. 1;

FIG. 3 is a sectional view taken through lines 3--3 of FIG. 2;

FIG. 4 is a sectional view of a portion of FIG. 2 taken through lines4--4;

FIG. 5 is an exploded perspective view of a portion of the embodiment ofFIG. 4;

FIG. 6 is an enlarged view, partly broken away, of a portion of theembodiment of FIG. 4;

FIG. 7 is a block diagram of a circuit that may be used for coordinationof a sensor, scanner drive and laser used in the embodiment of FIG. 9;

FIG. 8 is a perspective view from the top right side of anotherembodiment of scanning section usable in the DNA sequencing apparatus ofFIGS. 1 and 2;

FIG. 9 is a perspective view from the bottom right side of thesequencing system of FIG. 8;

FIG. 10 is a flow diagram of a program used to control the operation ofthe system of FIG. 8 and FIG. 9;

FIG. 11 is a more detailed flow diagram of the software control for aportion of the program of FIG. 10;

FIG. 12 is a schematic diagram of another portion of the program of FIG.10;

FIG. 13 is a block diagram of another embodiment of the program of FIG.12;

FIG. 14 is a block diagram of a control portion for the embodiments ofFIGS. 1-13;

FIG. 15 is a schematic diagram of a display arrangement useful in theembodiment of FIGS. 1-14; and

FIG. 16 is a front view of a display screen resulting from its use ofthe display arrangement of FIG. 15.

DETAILED DESCRIPTION

In FIG. 1, there is shown an embodiment 10 of sequencing system having acentral system 120, a plurality of remote stations, two of which areshown at 122A and 122B and a DNA fluorescent marking system 121. The DNAfluorescent marking system 121 includes means for labeling identicalstrands of DNA and a DNA preparation system. In this preparationprocess, strands are separated into four aliquots. The strands in agiven aliquot are either individually cleaved at or synthesized to anybase belonging to one or more of the four base types, which are adenine,guanine, cytosine and thymine (hereinafter A, G, C and T). The adenine-,guanine-, cytosine- and thymine-terminated strands are thenelectrophoresed for separation. The rate of electrophoresis indicatesthe DNA sequence.

The fluorescent markers are attached to the identical strands of morethan 100 bases in a container. The flourescent markers may be attachedto DNA primer molecules or to deoxynucleotide triphosphates used in thesynthesis of the DNA strands or to dideoxynucleotide triphosphates whichterminate synthesis of the DNA strands. Single or multiple fluorescentmarkers may be attached to the DNA fragments. They must be of such asize and have such chemical characteristics as to not obscure the normaldifferences in the mobilities between the different fragments due toterminations at different ones of the adenine, guanine, cytosine andthymine bases and be able to be easily detected.

The DNA fluorescent marking system 121 communicates with the centralsystem 120 as well as the remote stations 122A and 122B. The centralsystem 120 includes a separating system and a detection and processingsystem to separate the strands by length with each fragment beingterminated at a different one of the A, T, G and C groups. Theseparating system, which sequences strands by length, communicates withthe detection and processing system which analyzes the fragments bycomparison of the progress of each band of DNA fragments along the gelwith the other bands to derive information about the sequence of theDNA.

The separating system continuously sequences strands of DNA, and forthis purpose, the preferred embodiment includes at least fourelectrophoresis channels, each adapted to receive fluorescently labeledDNA strands having at one end a base of a given type. Each of thechannels has a gel path and electrical field across it identical in itscharacteristics to the gel path of the other channels and electricalfields across the other channels. The bands are detected in a mannerthat indicates their mobility in the gel to indicate the sequence of theA, G, C and T strands of different lengths.

The detection and processing system includes a scanning apparatus havinga light source, such as a laser or arc lamp or other suitable sourcethat emits light in the optimum absorption spectrum of the marker. Thelight may be split by the use of fiber. In the preferred embodiment, thelight source is a diode laser that irradiates the channels with infraredlight having a wavelength that matches the absorbance region of themarker. The detector includes a light sensor that is preferably anavalanche photodiode that is sensitive to the near infrared lightemission of the marker. It may include a filtering system having a passband suitable for passing selectively the optimum emission of thefluorescent marker to the light sensor.

The photodiode, photomultiplier or other light detector selectivelydetects the fluorescence using techniques which enhance the signal/noiseratio. One technique is to modulate the laser source by pulsing theelectrical current driving the laser and detect light in sequence withthe emitted light by connecting the output signal from the sensor incircuit with a lock-in amplifier that is sequenced with the pulsed laserlight. Another technique is the use of laser pulses which are less thanfive nanoseconds time duration, with detection in a time window. Thelength of such window and its delay from the pulse are optimized todiscriminate against background fluorescence as well as scattered laserlight.

To determine the sequence of strands, the processing system includesmeans for correlation between the channel in which the fluorescent lightis detected with the time of detection and means for indicating: (1) ifthe type of base termination or nucleotide cleavage is A, G, C or T; and(2) the time sequence of separation of each strand in each channel ofthe electrophoresis gel.

To use the apparatus to sequence DNA strands, identical DNA strands arenormally formed of a length greater than 100 bases. In one embodiment,the strands are marked by a suitable marker. The strands are dividedinto four aliquots and the strands within each aliquot are cleaved atany base belonging to a specific base type or are synthesized to anybase belonging to a specific base type. These four aliquots are thenelectrophoresed through identical channels to separate strands so thatthe shorter strands are resolved towards the end of the gel prior toresolution of the longer strands, which still are near the entrance endof the gel. In another embodiment, the strands are divided into fouraliquots and cleaved or synthesized to a given base before being marked.The same marker may be used for all four aliquots and separation may beperformed as described above or a different marker may be used for eachdifferent termination group of the A, T, C and G groups so as to processin a single channel for a complete sequence. This occurs in a continuousprocess so a substantial number of different length strands may beresolved in a relatively short gel. This methodology takes advantage oftime-resolved bands, as opposed to the limitations of spatial-resolvedbands.

The gel size, electric field and DNA mobilities are such that the moremobile bands are fully resolved while the less mobile bands are yetunresolved in a continuous process such that at least ten percent of thebands have been resolved by electrophoresis in the gel while the lessmobile bands which are near the entrance end of the gel are not fullyresolved. These less mobile bands become resolved little by little overtime in a continuous fashion without interruption of the movement ofthese bands through the gel. The markers are detected by transmittinginfrared light to fluorescently marked DNA strands which may be at thesame infrared wavelength or at four different infrared wavelengthsdepending on the embodiment of separation technique.

The remote stations 122A and 122B each are able to perform thesequencing but some portions of data processing can only be performed bythe central station 120. It may supply data to the remote stations, suchas 122A and 122B, to which it is electrically connected and receive datafrom them. With this arrangement, the central sequencing system 120 maycooperate with one or more of the remote stations, such as 122A and122B, for increased capability such as increased number of channels.Each unit may control the parameters used in sequencing, such as theelectrophoresis potential or the like.

In FIG. 2, there is shown a simplified view of the remote station 122Ahaving a cabinet housing 130, a front cover 132, a liquid crystaldisplay readout 134, a high voltage warning light 136 and a plurality offunction keys 138. In FIG. 2, the remote station 122A is shown closed.However, the front cover 132 may be removed to expose an electrophoresissection. The potential applied across the gel may be set and differentdata readouts may be selected either from the analysis provided withinthe central system 120 (FIG. 1) or values from within the remote station122A using the function key pad 138 and the selected data displayed onthe liquid crystal display readout 134 prior to and/or after selection.

In FIG. 3, there is shown a sectional view of a portion of the remotestation 122A taken through section lines 3--3 of FIG. 2 having anelectrophoresis section 140, a scanning section 142, an electrophoresispower supply 144, a system power supply section 144A, an analog board146 and a digital board 148. The electrophoresis section 140 ispositioned near the front of the cabinet and a portion of it is adaptedto be scanned by the scanning section 142 in cooperation with circuitryon the analog board 146 and the digital board 148. All of the apparatusare electrically connected to the power supply section 144A for suchoperation.

To separate different DNA fragments into bands, the electrophoresissection 140 includes a gel sandwich 150, an upper buffer assembly 152, asupport assembly 154, and a lower buffer assembly 151 positioned toenclose the bottom of the gel sandwich 150. In the embodiment of FIG. 3,the gel sandwich 150 is held substantially vertically and itstemperature is controlled during operation. Bands are separated byapplying voltage to the upper buffer assembly 152 and lower bufferassembly 151 and scanned by the scanning section 142.

To support the gel sandwich 150, the support assembly 154 includes apair of upper side brackets and lower side brackets 160 and 162 (onlyone of each pair being shown in FIG. 3), an apparatus support plate 168,a temperature control heating plate 164 and a plastic spacer, shown at166A-166C, in FIG. 3. The entire structure is supported on the apparatussupport plate 168 which mounts the upper and lower side brackets 160 and162.

The upper and lower side brackets 160 and 162 are each shaped to receivea pin such as 161 and 167 (FIG. 6) extending from a gel sandwich such asthe gel sandwich 150 and thus hold the gel sandwich in place on one sidein juxtaposition with the scanning section 142. The pin 167 (FIG. 6) onthe side of the sandwich opposite to the pin 161 (FIG. 6) fits into acorresponding one of two brackets 163 and 160 (FIG. 6) so that the gelsandwich can be hooked in place. The other two brackets 165 and 162 arepositioned to receive the pins of other length gel sandwiches with thelower bracket 162 receiving pins of shorter vertical length sandwichesthan the upper bracket 160. Even longer gel sandwiches can be mounted bysubstituting a longer heating plate for the heating plate shown at 164.

The spacer as shown as 166A-166C space the temperature control heatingplate 164 from the apparatus support plate 168 and maintain it at aconstant selected temperature above ambient temperature. In thepreferred embodiment, the temperature is maintained at 50 degreesCentigrade and should be maintained in a range of 30 degrees to 80degrees.

The scanning section 142 includes a laser diode assembly (not shown inFIG. 3), a microscope assembly 172, a photodiode section 174 and ascanner mounting section 176. The laser diode assembly (not shown inFIG. 3) is positioned at an angle to an opening in the apparatus supportplate 168 and the heating plate 164 so that light impinges on the gelsandwich 150 to cause fluorescence with minimum reflection back throughthe microscope assembly 172.

To receive the fluorescent light, the microscope assembly 172 is focusedon the gel sandwich 150 and transmits fluorescent light emittedtherefrom into the photodiode section 174 which converts it toelectrical signals for transmission to and processing by the analog anddigital boards 146 and 148 which may provide further analysis of data.The scanning section 142 moves along a slot in the apparatus supportplate 168 which is mounted to the scanner mounting section 176 duringthis operation in order to scan across the columns in the gel sandwich150.

The scanner mounting section 176 includes a mounting plate 180, abearing 182, a stepping motor 184, a slidable support 186 and a belt andpully arrangement 185, 188A and 188B. The mounting plate 180 is movablymounted to the apparatus support plate 168 through a frame member andsupports the elongated bearing plate 182, the stepping motor 184 and twopulleys 188A and 188B. The elongated bearing plate 182 extends thelength of the gel sandwich 150.

To permit motion of the laser diode assembly (not shown) and microscopeassembly 172 with respect to the gel sandwich 150, the slidable support186 supports the microscope assembly 172 and diode assembly and slidablyrests upon the bearing plate 182. An output shaft 183 of the steppingmotor 184 drives a pulley 188B through pulley 188, belt 185, and pulley188A and the pulley 188B drives a belt (not shown) that is clamped tothe slidable support 186 to move it the length of the gel sandwich 150during scanning by the laser diode and microscope assembly 172 whichrest upon it. The stepping motor 184 under the control of circuitry inthe digital board 148 moves the pulley 188B to move the belt (not shown)and thus cause scanning across the gel sandwich 150.

As shown in this view, the electrophoresis power supply 144 iselectrically connected to buffer in the upper buffer assembly 152through an electrical connector 194 and to the lower buffer assembly 151through a connector not shown in FIG. 3.

The upper buffer assembly 152 includes walls 197 forming a container tohold a buffer solution 195 and a cover 199 formed with a lip to fit overthe walls 197 from the top and containing a downwardly extending flatmember spaced away from the side walls and holding a conductor 211. Theconductor 211 is electrically connected to the source of power throughconnector 194 which is mounted to the top of the cover 199 to permitelectrical energization of the buffer solution 195.

The bottom buffer assembly 151 includes enclosed walls 201 defining acontainer for holding a buffer solution 203 and a cover 205 closing thecontainer 201 and having a downwardly extending portion 213 extendinginto the buffer solution 203 for supporting a conductor 207 for applyingenergy to the bottom buffer solution 203. The gel sandwich 150 extendsdownwardly into the buffer solution 203 and upwardly into the buffersolution 195 to permit the electrical contact for electrophoresis. An"O" ring 197B provides a seal for the upper buffer assembly 152 so thatthe buffer solution 195 does not empty out of the upper buffer assembly152.

In FIG. 4, there is shown a sectional view taken through lines 4--4 ofFIG. 2 showing a portion of the electrophoresis section 140, a portionof the scanning section 142 (indicated twice in FIG. 4 for clarity) andthe electrophoresis power supply section 144A (FIG. 3) mounted togetherto illustrate from a top view the arrangement of the apparatus supportplate 168, the heater plate 164, the gel sandwich 150, a laser diodeassembly 170, a microscope assembly 172 and a photodiode assembly 174.The heater plate 164 and apparatus support plate 168 have slots runningin a horizontal direction orthogonal to the lanes of DNA in theelectrophoresis section 140 sized to receive the ends of a laser diodeassembly 170 and the microscope assembly 172 for scanning thereof.

To cooperate with the separation and scanning of DNA bands, the gelsandwich 150 includes a front glass plate 200, a gel section 202 and arear glass plate 204 mounted in contact with the heater plate 164 andhaving a section exposed for scanning by the laser diode assembly 170and the microscope assembly 172. The rear glass plate 204 contacts theheater plate 164 and is separated from the front glass plate 200 by thegel section 202 within which DNA separation takes place. The front andrear glass plates 200 and 204 may be any type of glass but arepreferably soda lime which has low fluorescence in the infrared and nearinfrared regions and is prepared by a process that provides opticallyflat surfaces without grinding.

To transmit light to the gel sandwich 150, the laser diode assembly 170includes a housing 210, a focusing lens 212, a narrow band pass filter214, a collimating lens 216 and a laser diode 218. The laser diode 218emits infrared or near infrared light which is collimated by the lasercollimating lens 216 and filtered through the narrow band pass infraredfilter 214. This light is focused by the focusing lens 212 onto the gelsandwich 150. Preferably, the point of focus on the gel section 202 ofthe gel sandwich 150 lies along or near the central longitudinal axis ofthe microscope assembly 172 and the photodiode assembly 174.

The thickness of the glass plates and the gel, the position of the laserand microscope assembly and thus the angle of incidence and angle ofreflection of the light from the laser and to the microscope assemblyare chosen, taking into consideration the refractive index of the geland glass and the thickness of the glass plates and the gel, so that thelight from the laser is maximally transmitted to the gel. The light fromthe laser is not directly reflected back because the angle of incidenceto a normal is equal to the Brewster's angle at the first interface andis such as to impinge on the markers with full intensity afterrefraction but not be reflected by the first surface of the gel sandwich150 into the microscope assembly and the microscope assembly views thosemarkers that fluoresce in its line of sight.

To maintain temperature control over the laser diode, the housing 210:(a) is coupled to a heat sink through a thermal electric cooler 220, and(b) encloses the focusing lens 212, narrow band pass filter 214,collimating lens 216 and laser diode 218; and (c) accommodates theelectrical leads for the diode.

To receive and focus light emitted by fluorescent markers from the gelsection 202 in response to the light from the laser diode assembly 170,the microscope assembly 172 includes a collection lens 230, a housing232, and a focusing motor. The microscope assembly 172 is adapted to bepositioned with its longitudinal axis centered on the collection lens230 and aligned with the photodiode assembly 174 to which it isconnected. For this purpose, the housing 232 includes a centralpassageway in which are located one or more optical filters (not shown)with a pass band matching the emission fluorescence of the marked DNAstrands. With this arrangement, the collection lens 230 receives lightfrom the fluorescent material within the gel section 202 and collimatesthe collected light for optical filtering and then transmission to thephotodiode assembly 174.

To generate electrical signals representing the detected fluorescence,the photodiode assembly 174 includes a housing 240 having within it, asthe principal elements of the light sensors, an inlet window 242, afocusing lens 244, a sapphire window 246 and an avalanche photodiode248. To support the avalanche photodiode 248, a detector mounting plate250 is mounted within the housing 240 to support a plate upon which theavalanche photodiode 248 is mounted. The inlet window 242 fits withinthe housing 240 to receive light along the longitudinal axis of thephotodiode assembly 174 from the microscope assembly 172.

Within the housing 240 of the photodiode assembly 174, the sapphirewindow 246 and avalanche photodiode 248 are aligned along the commonaxis of the microscope assembly 172 and the photodiode assembly 174 andfocuses light transmitted by the microscope assembly 172 onto a smallspot on the avalanche photodiode 248 for conversion to electricalsignals. A thermoelectric cooler 252 utilizing the Peltier effect ismounted adjacent to the detector mounting plate 250 to maintain arelatively cool temperature suitable for proper operation of theavalanche photodiode 248.

As best shown in this view, the stepping motor 184 rotates the belt 185to turn the pulley 188A, which, in turn, rotates pulley 188B. The pulley188B includes a belt 177 extending between it and an idler pulley 179and is attached at one location to the slideable support 186 (FIG. 3) tomove the scanning microscope and laser lengthwise along the gel sandwich150 for scanning purposes. The motor 184, by moving the carriage backand forth accomplishes scanning of the gel sandwich 150.

In FIG. 5, there is shown a fragmentary perspective view of the gelsandwich 150 and the upper buffer assembly 152 mounted to each othershowing the outer glass plate 200 cut away from the rear glass plate 204to expose the gel section 202 to buffer solution within the upper bufferassembly 152. With this arrangement, DNA samples may be pipetted betweenthe glass plates 200 and 204 and moved downwardly by electrophoresisbeyond the upper buffer assembly 152 and through the gel sandwich 150 tothe bottom buffer (not shown in FIG. 5).

In FIG. 6, there is shown a broken away view of the gel sandwich 150illustrating the upper buffer assembly 152 and the lower buffer assembly151 connected to it at each end. As shown in this view, the cover 199includes a connecting post 214 which receives the elongated flexibleconductor 211 for connection to the downwardly extending portion of thecover 199 into the buffer compartment. This flexible conductor hassufficient length to accommodate different lengths of gel sandwichesthat cause the upper buffer assembly 152 to be at different elevations.In another embodiment, the length of conductor 211 is the same for thedifferent lengths of gel sandwiches in that this cover 199 is raised orlowered depending on the gel length. To accommodate electricalconnection in this embodiment, either the connecting post 214 mates withone of a series of mounting connectors located at different verticalpositions on the apparatus support plate 168 (FIG. 3) or the connectingpost 214 mates with a flexible extension cable that electricallyconnects the connecting post 214 with a mounting connector.

As best shown in this view, a plurality of side brackets 160, 163, 165and 169 are mounted to the apparatus support plate 168 (FIG. 3) toreceive pins 161 and 167 extending from the sides of the gel sandwich150 to support the gel sandwich in place. The pins 161 and 167 extendfrom opposite sides of the gel sandwich at the same elevation and thebrackets 160, 163, 165 and 169 are mounted in pairs to the apparatussupport plate 168 (FIG. 3) with each pair being at a different elevationand each bracket of a pair of brackets being positioned on the oppositeside of the gel sandwich from the other bracket of the same pair tosupport different sizes of gel sandwiches at a location that providesbalance to them.

The upper and lower side brackets 160 and 162 on one side of the gelsandwich and the upper and lower side brackets 163 and 165 on theopposite side are each shaped to receive a pin such as 161 and 167 (FIG.6) extending from the gel sandwich 150 and thus hold the gel sandwich inplace on one side in juxtaposition with the scanning section 142 (FIGS.3 and 4). The pin 167 on the side of the sandwich opposite to the pin161 fits into a corresponding one of the two brackets 163 and 165 sothat the gel sandwich can be hooked in place. For longer gel sandwiches,the pin 167 fits into bracket 163 while for shorter gel sandwiches thepin 167 fits into bracket 165. The other of the two brackets 160 and 169are positioned to receive the pin located on the opposite side of thegel sandwich such that the lower bracket 169 receives the pin of shortervertical length gel sandwiches and the upper bracket 160 receives thepin of longer vertical length gel sandwiches. Even longer gel sandwichescan be mounted by substituting a longer heater plate for the heaterplate shown at 164 (FIG. 3). As best shown in this view, recesses 231extend downwardly into the gel to receive DNA sample from a pipette andthus form channels for electrophoresing.

To form an electrical connection through the gel sandwich 150 from theupper buffer assembly 152 to the lower buffer assembly 151, a conductingpost 218 is connected to the cover 205 of the lower buffer assembly 151for receiving the conductor 207 (FIG. 3) which extends downwardly to thedownwardly extended plate 213 and into the buffer solution.

In FIG. 7, there is shown a block diagram of the circuitry used tocontrol the remote station 122A of the embodiment of FIG. 2 having acontrol, correlation and readout section 250, a scanner drive 176, themotor assembly 184 for moving the scanner drive 176, the sensingconfiguration 252 and the focusing motor assembly and pivot motorassembly controls 300 and 302 respectively.

The sensing configuration 252 includes the laser diode assembly 170 andthe photodiode assembly 174 which receives signals, removes some noise,and transmits the signals for display and read out in the control,correlation and read out section 250. At the same time, the scannerdrive 176 and motor for the scanner drive 184 receive signals from thecontrol, correlation and readout section 250 to control the motion ofthe sensor back and forth across the gel sandwich. This overallconfiguration is not part of the invention of this application exceptinsofar as it cooperates with the sensing configuration 252 to scan theDNA and determine its sequence in accordance with the embodiments ofFIGS. 1-6.

To drive the laser diode assembly 170 and the microscope assembly 172(FIGS. 3 & 4) and the photodiode assembly 174 from position to position,the motor assembly 184 includes a stepping motor 254 and a motor driver256. The motor drive 256 receives signals from the control correlationand readout section 250 and actuates the stepping motor 254 to drive thescanner drive 176. The scanner drive 176 is mechanically coupled to thestepping motor 254 through a belt and pulley arrangement for movementback and forth to irradiate and sense the electrophoresis channels onthe gel sandwich 150 (FIG. 3). The stepping motor 254 and motor driver256 are conventional and not themselves part of the invention.

To correlate the scan with received signals and provide a display ofthem, the control, correlation and readout system 250 includes acomputer 260 which may be any standard microprocessor, a televisiondisplay or cathode ray tube display 262 and a printer 264 for displayingand printing the results of the scans. Data, after being processed insensing configuration 252 is supplied to the computer 260 forcorrelation with the position of the scanner drive 176 as controlled bythe computer 260 and display 262.

To sense data, the sensing configuration 252 includes in addition to thelaser diode assembly 170 and the photodiode assembly 174, a choppercircuit 270, a sensor power supply 272, a preamplifier 274, a lock-inamplifier 276, a 6-pole filter 278, a 12-bit analogue digital converterinterface circuit 280 and a laser power supply 282. The photodiodeassembly 174 receives light from the laser diode assembly 170 after itimpinges upon the gel sandwich 150 (FIG. 3) and transmits electricalsignals through preamplifier 274 to the lock-in amplifier 276. Thephotodiode assembly 174 receives signals from the sensor power supply272. The chopper circuit 270 provides pulses at synchronized frequenciesto the lock-in amplifier 276.

The laser diode assembly 170 receives power from the power supply 282which is controlled by the chopper circuit 270 so that the signal fromthe laser diode assembly 170 is in synchronism with the signal appliedto the lock-in amplifier 276 so that the output from the lock-inamplifier 276 to the 6-pole filter 278 discriminates against unwantedsignal frequencies. This signal is converted to a digital signal in the12-bit analogue to digital converter 280 which serves as an interface tothe computer 260.

To maintain optical focus, the computer 260 is electrically connected tothe pivot motor assembly 302 and the focusing motor assembly 300, whichmotor assemblies are able to adjust the microscope assembly 172 (FIGS. 3and 4) to focus it and to adjust the location of the microscope withrespect to the gel sandwich 150 by pivoting the support bed 180 (FIGS. 4and 8). As will be better explained hereinafter, this permits a numberof different focusing arrangements such as one in which the microscopeis periodically refocused during an individual scan across the gel tomaintain focus and one in which the distance between the gel sandwichand the path of travel of the microscope is adjusted with respect toeach other so that during movement of the microscope as it scans, thefocal point is maintained on the gel even though there was originallysome non parallelism between the gel and the travel path of themicroscope along the course of a scan. Of course both motors may becontrolled simultaneously as better explained hereinafter.

In FIG. 8, there is shown a fragmentary, exploded top perspective viewof another embodiment of scanning section 142A substantially the same asthe scanning section 142 of FIGS. 3 and 4 in which the identical partsare indicated by the same reference numerals in each embodiment. Asshown in this view, the scanning section 142A includes three motorassemblies, the stepping motor assembly (scan motor assembly) 184, thefocusing motor 300 and a mounting plate pivot motor assembly 302 and apivot assembly 304. The mounting plate pivot motor assembly 302 is onlyin the embodiment of scanning assembly 142A but the focusing motor 300and stepping motor assembly 184 are in both the embodiment of scanningsection 142 and the embodiment 142A. The motor assembly 184 operates inthe same manner in both embodiments to drive the output shaft 183 (FIGS.3 and 9) which in turn drives the slidable support 186 (FIG. 3) on thebearing plate 182 (FIGS. 3 & 4) through the belt 185 (FIGS. 3 & 9) andtoothed belt 177.

The focusing motor assembly 300 is mounted for movement with themicroscope assembly 172 and photodiode assembly 174 to focus themicroscope assembly 172 directly into the gel to receive light directlyfrom the fluorescent markers therein. This focusing may be done manuallyor automatically by focusing at points where there is no fluorescentemission from DNA markers in the gel. This is done by sensing thefluorescence of the two glass plates which have relatively high emissionand causing the focusing to be between the two high emission glassplates and within the lower emission gel.

The pivot motor assembly 302 cooperates with the pivot assembly 304 toadjust the angle between the gel sandwich 150 (FIG. 3) and the mountingplate 180 so that the focus of the microscope assembly 172 can be setand the focal point remain within the gel section as the microscopeassembly 172 moves in a horizontal scanning direction across the gelsandwich 150 (FIG. 3). The pivot motor assembly 302 cooperates in thefocusing operation so that the microscope is focused at one point at oneend of the gel sandwich 150 and then the microscope assembly 172 scansacross to another widely separated point without changing the focus ofthe microscope lens.

After the microscope assembly 172 has moved to a new location, the pivotmotor assembly 302 then moves the support bed 180 about the pivot point305 with the motion being in a horizontal plane to adjust the angle inthe vertical plane of the support bed 180 and gel sandwich 150 withrespect to each other so that the plane of the gel sandwich 150 and thescan direction are parallel. The focus motor 300 then refocuses themicroscope assembly 172 so that the focus point is the same at bothextremes of a scan, thus permitting a continuous scan without the needto dynamically refocus the microscope. Of course either the gel sandwichor support for the microscope or both can be adjusted with minormodification of the equipment.

To permit adjustment of the horizontal scanning path of the microscopeassembly 172, the pivot assembly 304 in the preferred embodimentincludes an opening or eyelet at pivot point 305 with the pivot point305 having a vertical axis perpendicular to the support plate 180, acylindrical vertically oriented pivot pin 310 and a mounting housing 311rigidly mounted to the pivot pin 310. The pivot pin 310 fits rotatablywithin the support bed 180 and the support bed 180 is movably bolted tothe housing 311. The bolts (not shown in FIG. 8) have shanks that extendloosely through the openings 313 and 315 to permit movement between thesupport plate 180 and housing 311 to permit pivoting of the support bed180 with respect to the housing 311. The housing 311, which is mountedat end 317, and the corresponding end of a support member 306 aremounted to the apparatus support plate 168 (FIG. 3) to movably supportthe two ends of the support bed 180 on the DNA sequencer frame.

To provide pivoting, the pivot motor assembly 302 includes the motoroutput shaft 319, biasing member 321 and support member 306 so thatrotation of the motor 303 in one direction causes the motor output shaft319 to push against the vertical apparatus support plate 168 (FIG. 3) towhich it is movably mounted at one end to increase the angle, androtation in the other direction pulls it to reduce the angle or releasesit to be pulled by a biasing member 321 to move the bed forwardly to theplate 168 and decrease the angle. The support member 306 is mounted tothe support plate 180 by bolts (not shown in FIG. 8) having their shanksfitting through slots similar to 313 and 315 that are large enough topermit pivoting. The support member 306 is mounted to vertical apparatussupport plate 168 so that the support member 306 is supported to theframe of the DNA sequencer.

In FIG. 9, there is shown a bottom perspective view of the support plate180 showing a mounting support means 308, shaft 183, pivot hole 305 andbolt holes 313 and 315, the motor assembly 184, the pivot and the pivotmotor assembly 302 illustrating the manner in which the pivot pin ismounted to the frame to permit movement of the support plate 180 by thepivot motor assembly 302.

In the embodiment of FIGS. 8 and 9 the angle of the support plate 180 ischanged to cause the microscope to remain focused during a scan. Inanother embodiment, the angle of the support plate 180 is not changedbut the microscope is refocused at a number of points during ahorizontal scan to maintain the focus point within the gel. This methodhas certain inertia problems which slow down the scan or decrease itsprecision because of the larger number of times the assembly must startor stop.

In FIG. 10, there is shown a general block diagram 320 of a controlprogram permitting the computer to control the focusing of themicroscope during a scan so that the focal point remains within the gelof the gel sandwich. This program includes the general step 322 ofobtaining focus points such as a single right and left point or a numberof points and the step 324 of adjusting for scan depth and performingthe scan.

In one embodiment, the focus points are in the gel away from thechannels having DNA bands on both the right and left side of ahorizontal scan and in another embodiment, it is at a plurality ofpoints along a scan between channels having bands as well as near theends of the scans. In another embodiment, the focusing is performedprior to the DNA bands being electrophoresced so that it is notnecessary to select particular scan locations for the purpose ofavoiding such DNA bands. In the former embodiment, the microscope isfocused within the gel at one side, in which side there is the pivotpoint 305 (FIG. 8) in the scan support plate 180, and then at the secondpoint. The microscope is moved to the second point without changing thescan. When the microscope is at the second point, the support plate 180is moved so that, without adjusting the microscope further, the gelsandwich and support bed are altered in a parallel position. Then themicroscope is refocused such that the focal point is within the gel forboth locations, thus enabling a continuous scan thereafter which canproceed without dynamic focusing.

In the latter embodiment, as a scan is performed, the microscopeassembly 172 refocuses at a number of points, preferably refocusing thelens by means of the focusing motor assembly, although the focusingcould be done by adjusting the position of the support bed and gelsandwich with respect to each other or both focusing the lens and theposition of the support bed and gel plate with respect to each other.

In FIG. 11, there is shown a block diagram of a subsequence of substepswithin the step 322 for obtaining reference focal points including: (1)the substep 324 of beginning the program; (2) the substep 326 of movingthe microscope to the left side of the apparatus (the zero position) andturning the laser on; (3) the substep 328 of backing the focus motor up0.008 inches in 16 motor steps (the distance from microscope to gel isnow 0.008 inches beyond the starting point at the gel sandwich); (4) thesubstep 330 of causing the focusing motor assembly to move forward onestep, measuring the image signal one hundred times and obtaining theaverage of it; (5) the substep 332 of counting the steps the motor movesforward; (6) the substep 333 of deciding whether the count equals 32motions forward or not, returning to the step 330 if it does not and ifit does, the decision 332 to move to step 334; (7) the substep 334 ofcausing the focus motor to move back to its starting point; (8) thesubstep 336 of moving to a new position on the other side of theapparatus (right side in the preferred embodiment) at specified pointssuch as one inch increments up to six inches along the scan andrepeating steps 330 through 336 for each incremental location; and (9)followed by the decision step 336 of moving the scan back to itsoriginal zero point at end step 339.

With this arrangement, the microscope is moved to a starting point andfocused at a number of locations for that particular starting point,such locations including the gel within them as well as portions of theglass supporting plates. Data is taken at each location a multiplenumber of times and averaged for precision, with this data being stored.The microscope is then moved in a horizontal scan operation to anotherpoint and the process repeated so that at least two data points areobtained and stored in the memory of the computer. These data pointspermit the position of the microscope and gel to be adjusted withrespect to each other to maintain focus between them. It may benecessary to calculate the amount of pivoting of the microscope supportand gel about a pivot point if a measurement is not directly at thepivot point.

In FIG. 12, there is shown a block diagram illustrating a subsequencewithin the step 324 (FIG. 10) of adjusting for scan depth and performinga scan including: (1) the substep 340 of determining where the minimumlight omission occurred at different points; (2) the substep 342 ofhaving the focus and pivot points moved so that the microscope isfocused at the minimum fluorescent point at all of the locations; (3)the substep 344 of turning off the laser, moving the microscope to thezero position and resuming scanning with the laser turned back on ifdesired; and (4) the substep 346 of terminating the scan.

In FIG. 13, there is shown a block diagram of a program 350 illustratinga dynamic mode of scanning including: (1) the substep 352 of determiningthe minimum reading locations for left and right sides; (2) the substep354 of determining the position along an equidistant straight line forat least eight points; (3) the substep 356 of moving the microscope tothe zero position; (4) followed by the step 358 of scanning one section;(5) the step 360 of adjusting the focus again; (6) the step 362 ofcounting the adjustment; (7) the decision step 364 of determining if itis eight counts, and if not, returning to step 358 and repeating, and ifit is, ending the scan; and (8) substep 366 of ending the dynamicfocusing operation. With this arrangement, the microscope is refocusedeight times in a scan operation.

In FIG. 14, there is shown a program 370 for controlling the scancomprising: (1) the step 372 of beginning the scan; (2) the step 374 inwhich the customer uses the host computer or scanner keyboard to startscanning; (3) the step 375 in which the scanner software initializes DMA(direct memory access) pointers and initializes a final position in themotor control integrated circuit; (4) the step 376 in which the scanningsoftware tells the motor control integrated circuit to begin moving themicroscope; (5) the step 378 in which a traverse of the microscope each0.00048 inches causes the analog-to-digital converter to take onemeasurement and store that reading in memory; (6) the step 380 in whichthe scanner microprocessor is interrupted to say that a run is completedwhen the motor control integrated circuit is done moving a singleone-way trip, either left or right; (7) the step 382 of processing dataand sending it to the host computer while the motor is moving and takingdata, (8) the decision step 384 to determine if scanning is to becontinued, in which case the program returns to step 375 and if not,scanning is terminated at step 386; and (9) the step 386 of terminatingthe scan. As shown in this diagram, the computer control moves thescanner from place to place taking measurements along a path to performa scanning operation.

In FIG. 15, there is shown a block diagram of the display 400 having acathode ray tube 402, a horizontal scan circuit 404, a vertical scancircuit 406, an electron gun 408, a driver 410 and an intensity control412. As shown in this view, the horizontal scan control 404 periodicallyscans horizontally by applying a voltage to the deflection plates tomove an electron beam from the gun 408 horizontally across the screen ofthe tube 402. At a less rapid rate, the vertical control 406 changes thevertical voltage to deflect the electron beam and form a raster. Duringthe formation of the raster, data is applied to the driver 410 from thecomputer (FIG. 7) to modulate the voltage in the modulation control 412to change the intensity corresponding to data. As shown in FIG. 16, theface of the cathode ray tube 402 scans across with its data to form aplurality of bands 422 in which the existence of markers is shown by adifferent intensity of light on the screen so that DNA channels in thegel are shown as dark and light bands in a manner similar to that shownby gel electrophoresis. With this arrangement, the scanning rate may beset to discriminate against noise, particularly discriminating againstthe natural fluorescense of the glass in the gel sandwich 150 (FIGS. 3and 4). The screen display permits easy adjustment during measurementsor data retrieval.

From the above summary, it can be understood that the sequencingtechniques of this invention have several advantages, such as: (1) theytake advantage of resolution over time, as opposed to space; (2) theyare continuous; (3) they are automatic; (4) they are capable ofsequencing or identifying markers in relatively long strands includingstrands of more than 100 bases; and (5) they are relatively economicaland easy to use.

While in the preferred embodiment, a single emission frequency is usedin the infrared region in each channel and for all of A, T, G and Cterminated strands with the channel location identifying the terminatingbase type, multiple fluorescent markers can be used with the wavelengthbeing used to identify the base type. In such an embodiment, an opticalmeans detects a plurality of wavelengths and the computer correlatesintensity data, corresponding lanes and corresponding wavelengths.

Although a preferred embodiment of the invention has been described withsome particularity, many modifications and variations are possible inthe preferred embodiment within the light of the above description.Accordingly, within the scope of the appended claims, the invention maybe practiced other than as specifically described.

What is claimed is:
 1. A method for sequencing DNA comprising:applyingfluorescently marked DNA samples at a plurality of locations forelectrophoresing in a plurality of channels through a gelelectrophoresis slab; establishing electrical potential across said gelelectrophoresis slab wherein DNA samples are resolved in accordance withthe size of DNA fragments in said gel electrophoresis slab intofluorescently marked DNA bands; moving a scanning means with a carriageassembly a direction substantially perpendicular to a direction of theplurality of channels; and scanning the separated samplesphotoelectrically with a laser and a sensor mounted to the carriageassembly, wherein the laser scans with scanning light at a scanninglight frequency within an absorbent spectrum of said fluorescentlymarked DNA samples and sensing light at an emission frequency of themarked DNA; said step of scanning including the substeps of focusing amicroscope on a gel portion of an electrophoresis slab at least at twodifferent locations along a path of a single scan.
 2. A method forsequencing DNA comprising:applying fluorescently marked DNA samples at aplurality of locations for electrophoresing in a plurality of channelsthrough a gel electrophoresis slab; establishing electrical potentialacross said gel electrophoresis slab wherein DNA samples are resolved inaccordance with the size of DNA fragments in said gel electrophoresisslab into fluorescently marked DNA bands; and scanning the separatedsamples photoelectrically with a laser and a sensor, wherein the laserscans with scanning light at a scanning light frequency within anabsorbent spectrum of said fluorescently marked DNA samples and sensinglight at an emission frequency of the marked DNA; said step of scanningincluding the substeps of focusing a microscope on a gel portion of anelectrophoresis slab at least at two different locations along a path ofa single scan; said step of focusing at one location including the stepof adjusting the focal point of a lens of the microscope and the step offocusing at the other point including the step of adjusting the distancebetween the gel and the microscope.
 3. A method in accordance with claim2 in which said microscope is focused before taking data on a completescan and the distance between the microscope and said electrophoresisslab adjusted, after which a complete scan is made and data taken.
 4. Amethod for sequencing DNA comprising:applying fluorescently marked DNAsamples at a plurality of locations for electrophoresing in a pluralityof channels through a gel electrophoresis slab; establishing electricalpotential across said gel electrophoresis slab wherein DNA samples areresolved in accordance with the size of DNA fragments in said gelelectrophoresis slab into fluorescently marked DNA bands; and scanningthe separated samples photoelectrically with a laser and a sensor,wherein the laser scans with scanning light at a scanning lightfrequency within an absorbent spectrum of said fluorescently marked DNAsamples and sensing light at an emission frequency of the marked DNA;said step of scanning including the substeps of focusing a microscope ona gel portion of an electrophoresis slab at least at two differentlocations along a path of a single scan; the step of focusing in saidone location including the step of adjusting the focal point of a lensand the step of focusing at said at least one other location includingthe step of refocusing the lens at a distance no more than ten inchesfrom said first location, said steps of focusing including at least twoother further steps of focusing by adjusting the lens of the microscope.5. A method for sequencing DNA comprising:applying fluorescently markedDNA samples at a plurality of locations for electrophoresing in aplurality of channels through a gel electrophoresis slab; establishingelectrical potential across said gel electrophoresis slab wherein DNAsamples are resolved in accordance with the size of DNA fragments insaid gel electrophoresis slab into fluorescently marked DNA bands; andscanning the separated samples photoelectrically with a laser and asensor, wherein the laser scans with scanning light at a scanning lightfrequency within an absorbent spectrum of said fluorescently marked DNAsamples and sensing light at an emission frequency of the marked DNA:said step of scanning including the substeps of focusing a microscope ona gel portion of an electrophoresis slab at least at two differentlocations along a path of a single scan; the step of focusing includingthe step of locating a first and second glass plate by the amount offluorescence emitted by the glass plates and focusing on the gel betweenthe plates by detecting a different amount of fluorescence in the gel.6. A method in accordance with claim 5 in which the microscope isrefocused a plurality of times in each scan.
 7. A method for sequencingDNA comprising:applying fluorescently marked DNA samples at a pluralityof locations for electrophoresing in a plurality of channels through agel electrophoresis slab; establishing electrical potential across saidgel electrophoresis slab wherein DNA samples are resolved in accordancewith the size of DNA fragments in said gel electrophoresis slab intofluorescently marked DNA bands; and scanning the separated samplesphotoelectrically with a laser and a sensor, wherein the laser scanswith scanning light at a scanning light frequency within an absorbentspectrum of said fluorescently marked DNA samples and sensing light atan emission frequency of the marked DNA; said step of scanning includingthe substeps of focusing a microscope on a gel portion of anelectrophoresis slab at least at two different locations along a path ofa single scan to scan same, wherein a plurality of readings of data aretaken at a plurality of different points in a scan and those readingsaveraged.
 8. A method for sequencing DNA comprising:applyingfluorescently marked DNA samples at a plurality of locations forelectrophoresing in a plurality of channels through a gelelectrophoresis slab; establishing electrical potential across said gelelectrophoresis slab wherein DNA samples are resolved in accordance withthe size of DNA fragments in said gel electrophoresis slab intofluorescently marked DNA bands; and scanning the separated samplesphotoelectrically with a laser and a sensor, wherein the laser scanswith scanning light at a scanning light frequency within an absorbentspectrum of said fluorescently marked DNA samples and sensing light atan emission frequency of the marked DNA; said step of scanning includingthe substeps of focusing a microscope on a gel portion of anelectrophoresis slab at least at two different locations along a path ofa single scan to scan same, wherein data from said scan is directly usedto amplitude modulate density readings on a display; said scan isdisplayed in a vertical sweep of a cathode ray tube, whereby saidcathode ray tube provides intensity displays of bands representing DNA.9. A method for sequencing DNA comprising:applying fluorescently markedDNA samples at a plurality of locations for electrophoresing in aplurality of channels through a gel electrophoresis slab; establishingelectrical potential across said gel electrophoresis slab wherein DNAsamples are resolved in accordance with the size of DNA fragments insaid gel electrophoresis slab into fluorescently marked DNA bands; andscanning the separated samples photoelectrically with a laser and asensor, wherein the laser scans with scanning light at a scanning lightfrequency within an absorbent spectrum of said fluorescently marked DNAsamples and sensing light at an emission frequency of the marked DNA;said step of scanning including the substeps of focusing a microscope ona gel portion of an electrophoresis slab at least at two differentlocations along a path of a single scan, wherein said scanning means isaligned with said gel and the bands are scanned while still in the gelbut after being resolved.
 10. A method for sequencing DNAcomprising:applying fluorescently marked DNA samples at a plurality oflocations for electrophoresing in a plurality of channels through a gelelectrophoresis slab; establishing electrical potential across said gelelectrophoresis slab wherein DNA samples are resolved in accordance withthe size of DNA fragments in said gel electrophoresis slab intofluorescently marked DNA bands; scanning the separated samplesphotoelectrically with a laser and a sensor, wherein the laser scanswith scanning light at a scanning light frequency within an absorbentspectrum of said fluorescently marked DNA samples and sensing light atan emission frequency of the marked DNA; said step of scanning includingthe substeps of focusing a microscope on a gel portion of anelectrophoresis slab at least at two different locations along a path ofa single scan, wherein said scanning means is aligned with said gel andthe bands are scanned while still in the gel but after being resolved;and resolving the bands within at least one channel so that the bands ofthe more mobile strands in the channel are fully resolved while some ofthe less mobile strands to be later formed into bands are unresolved ina continuous process such that at least ten percent of the bands arefully resolved while the less mobile strands are yet unresolved intobands in the channel.
 11. A method of DNA sequencing comprising thesteps of:applying opposite polarity electrical potentials to a first andat least a second buffer; applying fluorescently marked DNA strands to aplurality of channels of gel, whereby said fluorescently marked DNAstrands are electrophoresed along said gel so that the bands of moremobile strands in at least one channel are fully resolved while some ofthe less mobile strands to be later formed into bands are unresolved ina continuous process; focusing a microscope wherein the focus point ofthe microscope is within the gel during a scan; moving the microscopeand laser across said channels, wherein light emitted from the laser andsaid microscope scans across said channels; and detecting fluorescentlight emitted by said fluorescently marked DNA strands, whereby the timesequence of separated bands may be obtained.
 12. A method of DNAsequencing comprising the steps of:applying opposite polarity electricalpotentials to a first and at least a second buffer; applyingfluorescently marked DNA strands to a plurality of channels of gel,whereby said fluorescently marked DNA strands are electrophoresed alongsaid gel so that the bands of more mobile strands in at least onechannel are fully resolved while some of the less mobile strands to belater formed into bands are unresolved in a continuous process; focusinga microscope wherein the focus point of the microscope is within the gelduring a scan; and scanning across said channels with light emitted froma laser and with said microscope; detecting fluorescent light emitted bysaid fluorescently marked DNA strands, whereby the time sequence ofseparated bands may be obtained, wherein light from said laser is in theband incorporating at least the near infrared and infrared regions andsaid detector responds to light in a band including at least said nearinfrared and infrared regions.
 13. A method of DNA sequencing comprisingthe steps of:applying opposite polarity electrical potentials to a firstand at least a second buffer; applying fluorescently marked DNA strandsto a plurality of channels of gel, whereby said fluorescently marked DNAstrands are electrophoresed along said gel so that the bands of moremobile strands in at least one channel are fully resolved while some ofthe less mobile strands to be later formed into bands are unresolved ina continuous process; focusing a microscope wherein the focus point ofthe microscope is within the gel during a scan; scanning across saidchannels with light emitted from a laser and with said microscope; anddetecting fluorescent light emitted by said fluorescently marked DNAstrands, whereby the time sequence of separated bands may be obtained;the step of focusing said microscope including the step of adjusting thedistance between said microscope and said gel wherein the distance doesnot vary more than five percent during a scan.
 14. A method of DNAsequencing comprising the steps of:applying opposite polarity electricalpotentials to a first and at least a second buffer; applyingfluorescently marked DNA strands to a plurality of channels of gel,whereby said fluorescently marked DNA strands are electrophoresed alongsaid gel so that the bands of more mobile strands in at least onechannel are fully resolved while some of the less mobile strands to belater formed into bands are unresolved in a continuous process; focusinga microscope wherein the focus point of the microscope is within the gelduring a scan; and scanning across said channels with light emitted froma laser and with said microscope; detecting fluorescent light emitted bysaid fluorescently marked DNA strands, whereby the time sequence ofseparated bands may be obtained; the step of focusing including the stepof dynamically refocusing at a plurality of locations to maintain thefocal point within an area of low radiation between two layers of higherradiation whereby said focal point is in the gel between two glassplates on either side of the gel.
 15. A method of DNA sequencingcomprising the steps of:applying opposite polarity electrical potentialsto a first and at least a second buffer; applying fluorescently markedDNA strands to a plurality of channels of gel whereby said fluorescentlymarked DNA strands are electrophoresed along said gel so that the bandsof more mobile strands in at least one channel are fully resolved whilesome of the less mobile strands to be later formed into bands areunresolved in a continuous process; focusing a microscope wherein thefocus point of the microscope is within the gel during a scan; scanningacross said channels with light emitted from a laser and with saidmicroscope; and detecting fluorescent light emitted by saidfluorescently marked DNA strands, whereby the time sequence of separatedbands may be obtained, wherein the step of focusing includes the step offocusing at one location; moving the microscope along a scan to anotherlocation; and adjusting the distance between the microscope and the geluntil the microscope can remain focused during at least a portion of thescan.
 16. A method of DNA sequencing comprising the steps of:applyingopposite polarity electrical potentials to a first and at least a secondbuffer; applying fluorescently marked DNA strands to a plurality ofchannels of gel, whereby said fluorescently marked DNA strands areelectrophoresed along said gel so that the bands of more mobile strandsin at least one channel are fully resolved while some of the less mobilestrands to be later formed into bands are unresolved in a continuousprocess; focusing a microscope wherein the focus point of the microscopeis within the gel during a scan; scanning across said channels withlight emitted from a laser and with said microscope; detectingfluorescent light emitted by said fluorescently marked DNA strands,whereby the time sequence of separated bands may be obtained;transmitting intensity data to a cathode ray tube so that the intensitydata extends substantially across a horizontal sweep of the cathode raytube; modulating an electron gun of the cathode ray tube with saidintensity data during horizontal sweeps; and spacing said horizontalsweeps vertically to present a plurality of bands.
 17. Apparatus forsequencing DNA comprising:means for applying fluorescently marked DNAsamples at a plurality of locations for electrophoresing in a pluralityof channels through a gel electrophoresis slab; means for establishingelectrical potential across said gel electrophoresis slab wherein DNAsamples are resolved in accordance with the size of DNA fragments insaid gel electrophoresis slab into fluorescently marked DNA bands; andmeans for scanning the separated samples photoelectrically with a laserand a sensor, wherein the laser scans with scanning light at a scanninglight frequency within an absorbent spectrum of said fluorescentlymarked DNA samples as the sensor and scanner move in a directiontransverse to the channels; said sensor including means for sensinglight at an emission frequency of the marked DNA; and said means forscanning including means for focusing a microscope on the gel portion ofthe electrophoresis slab at least at two different locations along thepath of a single scan.
 18. Apparatus for sequencing DNA comprising:meansfor applying fluorescently marked DNA samples at a plurality oflocations for electrophoresing in a plurality of channels through a gelelectrophoresis slab; means for establishing electrical potential acrosssaid gel electrophoresis slab wherein DNA samples are resolved inaccordance with the size of DNA fragments in said gel electrophoresisslab into fluorescently marked DNA bands; and means for scanning theseparated samples photoelectrically with a laser and a sensor, whereinthe laser scans with scanning light at a scanning light frequency withinan absorbent spectrum of said fluorescently marked DNA samples andsensing light at an emission frequency of the marked DNA; said means forscanning including means for focusing a microscope on the gel portion ofthe electrophoresis slab at least at two different locations along thepath of a single scan; said means for focusing at one location includingmeans for adjusting the focal point of the lens of a microscope andmeans for adjusting the distance between the gel and the microscope. 19.Apparatus in accordance with claim 18 in which the means for focusing insaid one location includes means for adjusting the focal point of thelens and the means for focusing at said at least one other locationincludes means for refocusing the lens at a distance no more than teninches from said first location, wherein the lens of the microscope maybe adjusted at least two further times to focus the microscope during ascan.
 20. Apparatus in accordance with claim 18 wherein said means forfocusing includes means for focusing before taking data on a completescan and the distance between the microscope and a plate adjusted, afterwhich a complete scan is made and data taken.
 21. Apparatus forsequencing DNA comprising:means for applying fluorescently marked DNAsamples at a plurality of locations for electrophoresing in a pluralityof channels through a gel electrophoresis slab; means for establishingelectrical potential across said gel electrophoresis slab wherein DNAsamples are resolved in accordance with the size of DNA fragments insaid gel electrophoresis slab into fluorescently marked DNA bands; andmeans for scanning the separated samples photoelectrically with a laserand a sensor wherein the laser scans with scanning light at a scanninglight frequency within an absorbent spectrum of said fluorescentlymarked DNA samples and sensing light at an emission frequency of themarked DNA; said means for scanning including means for focusing amicroscope on the gel portion of the electrophoresis slab at least attwo different locations along the path of a single scan; the means forfocusing including means for locating a first and second glass plate bythe amount of fluorescence emitted by the glass plates and focusing onthe gel between the plates by detecting a different amount offluorescence in the gel.
 22. Apparatus in accordance with claim 21 inwhich the microscope is refocused a plurality of times in each scan. 23.Apparatus for sequencing DNA comprising:means for applying fluorescentlymarked DNA samples at a plurality of locations for electrophoresing in aplurality of channels through a gel electrophoresis slab; means forestablishing electrical potential across said gel electrophoresis slabwherein DNA samples are resolved in accordance with the size of DNAfragments in said gel electrophoresis slab into fluorescently marked DNAbands; means for scanning the separated samples photoelectrically with alaser and a sensor, wherein the laser scans with scanning light at ascanning light frequency within an absorbent spectrum of saidfluorescently marked DNA samples and sensing light at an emissionfrequency of the marked DNA; said means for scanning including means forfocusing a microscope on the gel portion of the electrophoresis slab atleast at two different locations along the path of a single scan; andmeans for obtaining a plurality of readings of data that are taken at aplurality of different points in a scan and those readings averaged. 24.Apparatus for sequencing DNA comprising:means for applyinq fluorescentlymarked DNA samples at a plurality of locations for electrophoresing in aplurality of channels through a gel electrophoresis slab; means forestablishing electrical potential across said gel electrophoresis slabwherein DNA samples are resolved in accordance with the size of DNAfragments in said gel electrophoresis slab into fluorescently marked DNAbands; means for scanning the separated samples photoelectrically with alaser and a sensor, wherein the laser scans with scanning light at ascanning light frequency within an absorbent spectrum of saidfluorescently marked DNA samples and sensing light at an emissionfrequency of the marked DNA; said means for scanning including means forfocusing a microscope on the gel portion of the electrophoresis slab atleast at two different locations along the path of a single scan; andmeans for directly using the data from said scan to amplitude modulatedensity readings on a display, wherein said scan is displayed in avertical sweep of a cathode ray tube, whereby said cathode ray tubeprovides intensity displays of bands representing DNA.
 25. Apparatus forsequencing DNA comprising:means for applying fluorescently marked DNAsamples at a plurality of locations for electrophoresing in a pluralityof channels through a gel electrophoresis slab; means for establishingelectrical potential across said gel electrophoresis slab wherein DNAsamples are resolved in accordance with the size of DNA fragments insaid gel electrophoresis slab into fluorescently marked DNA bands; andmeans for scanning the separated samples photoelectrically with a laserand a sensor, wherein the laser scans with scanning light at a scanninglight frequency within an absorbent spectrum of said fluorescentlymarked DNA samples and sensing light at an emission frequency of themarked DNA; said means for scanning including means for focusing amicroscope on the gel portion of the electrophoresis slab at least attwo different locations along the path of a single scan, wherein saidscanning means is aligned with said gel and the bands are scanned whilestill in the gel but after being resolved.
 26. Apparatus in accordancewith claim 25 further including means for resolving the bands within atleast one channel so that the bands of more mobile strands in thechannel are fully resolved while some of the less mobile strands to belater formed into bands are unresolved in a continuous process such thatat least ten percent of the bands are fully resolved while the lessmobile strands are yet unresolved into bands in the channel. 27.Apparatus of DNA sequencing comprising:means for applying oppositepolarity electrical potentials to a first and at least a second buffer;means for applying fluorescently marked DNA strands to a plurality ofchannels of gel, whereby said fluorescently marked DNA strands areelectrophoresed along said gel so that the bands of more mobile strandsin at least one channel are fully resolved while some of the less mobilestrands to be later formed into bands are unresolved in a continuousprocess; means for focusing a microscope wherein the focus point of themicroscope is within the gel during a scan; means for scanning acrosssaid channels with light emitted from a laser and with said microscope;and means for detecting fluorescent light emitted by said fluorescentlymarked strands, whereby the time sequence of separated bands may beobtained.
 28. Apparatus for DNA sequencing comprising:means for applyingopposite polarity electrical potentials to a first and at least a secondbuffer; means for applying fluorescently marked DNA strands to aplurality of channels of gel, whereby said fluorescently marked DNAstrands are electrophoresed along said gel so that the bands of aremobile strands in at least one channel are fully resolved while some ofthe less mobile strands to be later formed into bands are unresolved ina continuous process; means for focusing a microscope wherein the focuspoint of the microscope is within the gel during a scan; means forscanning across said channels with light emitted from a laser and withsaid microscope; and means for detecting fluorescent light emitted bysaid fluorescently marked strands, whereby the time sequence ofseparated bands may be obtained, wherein light from said laser is in theband incorporating at least the near infrared and infrared regions andsaid detector responds to light in a band including at least said nearinfrared and infrared regions.
 29. Apparatus for DNA sequencingcomprising:means for applying opposite polarity electrical potentials toa first and at least a second buffer; means for applying fluorescentlymarked DNA strands to a plurality of channels of gel, whereby saidfluorescently marked DNA strands are electrophoresed along said gel sothat the bands of more mobile strands in at least one channel are fullyresolved while some of the less mobile strands to be later formed intobands are unresolved in a continuous process; means for focusing amicroscope wherein the focus point of the microscope is within the gelduring a scan; means for scanning across said channels with lightemitted from a laser and with said microscope; and means for detectingfluorescent light emitted by said fluorescently marked strands, wherebythe time sequence of separated bands may be obtained; the means forfocusing said microscope including means for adjusting the distancebetween said microscope and said gel wherein the distance does not varymore than five percent during a scan.
 30. Apparatus for DNA sequencingcomprising:means for applying opposite polarity electrical potentials toa first and at least a second buffer; means for applying fluorescentlymarked DNA strands to a plurality of channels of gel, whereby saidfluorescently marked DNA strands are electrophoresed along said gel sothat the bands of more mobile strands in at least one channel are fullyresolved while some of the less mobile strands to be later formed intobands are unresolved in a continuous process; means for focusing amicroscope wherein the focus point of the microscope is within the gelduring a scan; means for scanning across said channels with lightemitted from a laser and with said microscope; and means for detectingfluorescent light emitted by said fluorescently marked strands, wherebythe time sequence of separated bands may be obtained the means forfocusing including means for dynamically refocusing at a plurality oflocations to maintain the focal point within an area of low radiationbetween two layers of higher radiation whereby said focal point is inthe gel between two glass plates on either side of the gel. 31.Apparatus for DNA sequencing comprising:means for applying oppositepolarity electrical potentials to a first and at least a second buffer;means for applying fluorescently marked DNA strands to a plurality ofchannels of gel, whereby said fluorescently marked DNA strands areelectrophoresed along said gel so that the bands of more mobile strandsin at least one channel are fully resolved while some of the less mobilestrands to be later formed into bands are unresolved in a continuousprocess; means for focusing a microscope wherein the focus point of themicroscope is within the gel during a scan; means for scanning acrosssaid channels with light emitted from a laser and with said microscope;and means for detecting fluorescent light emitted by said fluorescentlymarked strands, whereby the time sequence of separated bands may beobtained; the means for focusing including means for focusing at onelocation; moving the microscope along a scan to another location; andadjusting the distance between the microscope and the gel so that themicroscope is focused on the gel during at least a portion of a scan.32. Apparatus for DNA sequencing comprising:means for applying oppositepolarity electrical potentials to a first and at least a second buffer;means for applying fluorescently marked DNA strands to a plurality ofchannels Of gel, whereby said fluorescently marked DNA strands areelectrophoresed along said gel so that the bands of more mobile strandsin at least one channel are fully resolved while some of the less mobilestrands to be later formed into bands are unresolved in a continuousprocess; means for focusing a microscope wherein the focus point of themicroscope is within the gel during a scan; means for scanning acrosssaid channels with light emitted from a laser and with said microscope:and means for detecting fluorescent light emitted by said fluorescentlymarked strands, whereby the time sequence of separated bands may beobtained; means for transmitting intensity data to a cathode ray tube sothat the intensity data extends substantially across a horizontal sweepof the cathode ray tube; means for modulating an electron gun of thecathode ray tube with said intensity data; and means for spacing saidhorizontal sweeps vertically to present a plurality of bands.